SmartFlare live RNA detection

I recently came across a booklet regarding SmartFlare for live cell RNA level detection. The basic idea, if i understand it correctly, is the use of gold nanoparticles that are conjugated to fluorescently tagged short double stranded probes. The “capture” strand is bound to the gold particle, is non-fluorescent and complementary to the target mRNA. The “reporter” strand is complementary to the capture strand but shorter. It is fluorescently labeled, but the gold is quenching the fluorescence.

This particle can enter the cells and if the target RNA is present, it replaces the “reporter” strand which is set free to the cytoplasm and fluoresce. This fluorescent can then be detected by microscopy or any other fluorescent detection.

SmartFlare concept. Source: Merck Millipore website

I haven’t used it yet because it clearly isn’t suited for single-molecule analysis. Furthermore, is does not give any data on RNA localization since the fluorescence appears only after the target RNA is bound and the “reporter” is a short oligo which probably diffuses very fast.

Furthermore, I’m concerned about the biological effects. Although they claim in their FAQ section that at the concentrations used it does not cause RNA knockdown and is non-toxic, I am concerned first about the short reported oligo which presumably will hybridize with antisense to the target RNA, if present and second, that capturing the target RNA will affect its gene expression (probably prevent its translation, maybe mis-localize it and can promote its decay thus actually affect its transcription rate as well).

Particularly I’m concerned due to the long incubation time they suggest in their protocol – 18 hours. Why so long? Shouldn’t it work at shorter time-scales?

This system can be a good reporter for relative expression levels for whole cell populations or single-cell analysis, but I’m hesitant about the use of these same cells for downstream assays.

Also, their illustrations always show complementary of the capture strand to the 5′ end of the target RNA. Does it have to be the 5′ end?

I am considering the use of this system as a reporter for my experimental purposes. Any thoughts? Did anyone use that and can share their experience?

8 responses to “SmartFlare live RNA detection”

The SmartFlare approach is very susceptible to off-target effects. The Mirkin lab (who invented the technology) has published an article http://www.ncbi.nlm.nih.gov/pubmed/24841494
showing that the fluorescent oligo/reporter strand detaches from the gold particle in the endosome. So the signal appears before it can reach its mRNA target.

Gal-
Great article and thank you for considering SmartFlare. I’d appreciate the opportunity to discuss SmartFlare with you in more detail to clarify some points (the Mirkin paper Luke is referring to is a different variant of the nanoparticle – different architecture (single strands), different conditions (higher concentration) and isn’t entirely representative of SmartFlare performance). A better paper you should see (out of the same Mirkin lab) is one where SmartFlares (aka NanoFlares) were used with CTCs: http://www.ncbi.nlm.nih.gov/pubmed/25404304

The fate of the messenger is pre-determined

Meta

Disclaimer

The opinions expressed in this blog are solely those of the author. Any advice given in this blog is based on the author's knowledge and experience, which may be lacking. Therefore, the author of this blog may be wrong (don't tell his wife!).
Images are used here for research and criticism in accordance with fair use policies.